Preparation of proteolytic enzymes having maximum activity at high alkalinity

ABSTRACT

A method of making proteolytic enzyme preparations having maximum activity at high alkalinity by submerged aerobic cultivation of strains of Bacillus alcalophilus in a nutrient medium containing assimilable carbon and nitrogen sources, while maintaining the pH-value of the nutrient medium between 7.5 and 11.

United States Patent 1151 3,674,643

Aunstrup et al. 1 July 4, 1972 54] PREPARATION OF PROTEOLYTIC 53 Field 61 Search ..l95/66 R, 62

ENZYMES HAVING MAXIMUM ACTIVITY AT HIGH ALKALINITY [561 [72] Inventors: Knud Aunstrup, Farum; Otto Andresen, UNITED STATES PATENTS p g Belle p, Vaerlose, all 2,530,210 11/1950 Smythe et al ..l9S/66 UX of Denmark [73] Assignee: Novo Terapeutisk Laboratorlum AIS, Sfgifjglfiihggifiggf;

Copenhagen Denmark Attorney-Synnestvedt & Lechner [22] Filed: Nov. 1, 1968 57 ABSTRACT [2]] App]. No.: 772,830 l A method of making proteolytic enzyme preparations having maximum activity at high alkalinity by submerged aerobic culfl elgn Application Priority Data tivation of strains of Bacillus alcalaphilu: in a nutrient medium containing assimilable carbon and nitrogen sources, while Nov. 10, 1967 Great Br1ta1n .5 l ,307/6' I maintaining the pH va]ue of the nutrifim medium between 75 d l l. 52 us. 01. ..l/62, /66 R an 3 Claims, No Drawings Int. Cl. .1C12d 11/00, Cl2d 13/10 PREPARATION OF PROTEOLYTICENZYMES HAVING MAXIMUM ACTIVITY AT HIGH ALKALINITY The present invention relates to a process of making novel proteolytic enzyme preparations for industrial use.

it is known that a number of bacteria produce proteolytic enzymes during their metabolism, and some of these bacteria have been cultivated on an industrial scale for the purpose of recovering the proteolytic enzymes produced.

In the Dutch periodical Antonie van Leeuwenhoek, 1, 141-147 (1934), A; Vedder described his experiments to isolate certain bacteria from feces. By enrichment in. alkaline peptone water and cultivation on what he called. glycocoll plates containing hemoglobin, KOH, glycocoll and peptoneagar, or better "carbonate plates" containing hemoglobin, K,CO KHCO, and peptone agar, Vedder, succeeded in isolating 16 strains of a bacterium belonging to the genus Bacillus, and being considered by Vedder to belongto a novel species which he called Bacillus alcalophilus because this species did only grow at pH values above 7, preferablywithin the range of 8.6 to 10. This species Bacillus alcalaphilus was incorporated in the 6th edition of Bergeys Manual of Deter'minative Bacteriology, but not inthe 7th edition.

One strain of Bacillus alcalophilus was deposited by Vedder with National Collection of Type Cultures in London under the NCTC-number 4553 and the same strain was later in 1956 deposited with National Collection of Industrial: Bacteria in Edinburgh under the NCIB' number 8772, where the strain by a mistake is considered as a Bacillus subtilis.

As the species alcalophilus was first isolated by Vedder, it will in the following be called Bacillus alcalophilus Vedder.

It has now been found that during their metabolism the strains of the species Bacillus alcalaphilus Vedder produce novel proteolytic enzymes e'xtracellularly, that such enzymes may be recovered from the cultivation media and that they show a pronounced activity at.high alkalinities, which makes themuseful within various industrial fields.

The process according to the invention is characterized by cultivating a strain of Bacillus alcalophilus Vedder under submerged aerobic conditions in a nutrient medium. containing assimilable carbon and nitrogen sources, maintaining the pH value of the nutrient medium between 7.5 and ll and recovering the proteolytic enzyme produced extracellularly during the cultivation. 1

The nutrient medium is composed in agreement with the principles of the known art. Suitable assimilable carbon.

sources are carbohydrates, such as saccharose, glucose,

starch, cereal grains, malt, rice, sorghum etc. The carbohydrate concentration may vary within rather wide limits, e.g., up to percent and down to 1-5 percent, but usually 8-10 percent would be suitable, the percentage bein calculated as dextrose. It has been found that the presence in the nutrient medium of carbohydrates will give rise to the formation of acidic components, resulting in a decreaseof the pH value during the cultivation. As it is essential to maintain a pH value of the nutrient medium within the range of 7.5 to 11, preferably 8 to 10, during the cultivation, measurements should be taken that the pH value does notfall below that range for any essential periodduringthe cultivation. lnorder to keep the pH value within the'required range,.a limited amount of carbohydrates may be used together with a buffer substance which is able to maintain the required pH value. Carbonates, and particularly sesquicarbonates, used in a concentration of up to 0.2 M in the medium, are able to create a pH-value of about 9.3 to 10.5, respectively.

Also other bufier systems, such as phosphate buffers, may

be used.

It is also-possible to initiate the cultivation with a low carbohydrate content and to add small amounts of carbohydrates successively during the cultivation.

A third possibility is to make use of automatic pH control by.

addition of various basic-reacting substances used'in this art.

The use of carbonates and aesquicarbonates as pHcontrolling substancesis very useful and itissurprising that itis possible during the cultivation on an industrial scale to use these compounds in theconcentrations referred to.

The nitrogen source in the nutrient medium may be of inorganic and/or organic nature. Suitable inorganic nitrogen sources are nitrates and ammonium salts, and among the organic nitrogen sources there are quite a number known for use in fermentation processes andin the cultivation of bacteria. [1- lustrating examples are soybean meal, cotton seed meal, peanut meal, casein, corn steep liquor, yeast extracts, urea, albumine etc.

Besides, the nutrient medium should contain the usual trace substances.

The temperature at which the cultivation takes place is normally within the same range as in the known cultivation of known species of the genus Bacillus. Usually a temperature between 25 and 40 C is convenient. The temperature is preferably 30 to 37 C.

As the cultivation has to be carried out under aerobic conditions, it is, when using fermentation tanks, necessary to make use of artificial aeration. The amount of air is similar to that used in the known cultivation processes.

In general, maximum yields of the proteolytic enzymes will be obtained after a cultivation time of l to 5 days.

For cultivation with proteolytic enzyme production and recovery in view, use has been made of the deposited strain from both of the collections referred to above, viz, strain NCl C 4553 and NCIB 8772. These strains show optimal growth at a pH value within the range of 7.8 and 9.0. The cultivation has been carried out bothin shake flasks and in pilot plant fermentors with artificial aeration. The yields obtained have been determined by the well-known Anson hemoglobin method, cfr. Journal of General Physiology, 22, 79-98 (.1939). One Anson unit means throughout this specification the amount of proteolytic enzyme digesting hemoglobin at a pH value of 10.1 and a temperature of 25 C during a reaction time of 10 minutes with such an initial'velocity that per minute there is formed an amount of split products which cannot be precipitated" with trichloracetic acid that these split products give the same color with phenol reagent as does one milliequivalent of tyrosine.

For cultivation the following two media were used:

1. Medium BPFA with the following composition:

Sodium caseinate Na HPO, 12H,0

10 g per liter of tap water 9 g per liter of tap water 2. Medium BSX with the follow composition:

Barley flour Soybean meal g per liter of tap water 30 g per liter of tap water Both these media were adjusted to the desired pH value by the addition of sesquicarbonate or soda under sterile conditions.

The experiments in shake flasks were carried out in 500 mm shake flasks, each of the flasks containing 100 ml of the nutrient medium BPFA and BSX, respectively, which were sterilized beforehand by autoclaving for 90 minutes at C, and after the autoclaving the pH value was adjusted to 9.3l0.3 by addition of sodium carbonate or sodium sesquicarbonate in aconcentration of 0.2 M and 0.1 M, respectively. There were used four flasks for each bacterium, and samples from the culture media for determining the enzyme content expressed inAnson units were taken after cultivation in 3, 4, 5, and 6 days, respectively. The flasks were during ,the cultivation placed one rotating table with 220 revolutions per minute, and the temperature during the cultivation was 30 C.

The maximum proteolytic activity measured in Anson units per kg of the nutrient medium appears from Table 1 below.

TABLE I Bacillus Maximum proteoalcalophi- Nutrient lytic activity Final pH lus Vedder medium Anson units per kg value NCIB 8772 BPFA 14 7.7 25 7.7 BSX 6 8.8 2.5 8.2 NCTC 4553 BPFA 16 7.9 13 7.6

By fractionated precipitation with ethyl alcohol enzyme preparations in powder form were prepared as it appears from the following Table 11.

The strain NCIB 8772 has also been cultivated in 550 liters stainless steel tanks under submerged conditions and artificial aeration while using 250 liters of the nutrient medium BPFA referred to above. The pH value of the medium was adjusted on 10.2. by sterile addition of 15 liters of 2 M soda solution. The temperature during the cultivation was 30 C, the velocity of the agitator 570 rpm and the aeration 0.25 m air per minute. After a cultivation period of 110 hours the pH value of the cultivation broth was 8.9 and the proteolytic activity was 17 Anson units per kg, measured at pH 10.1.

The enzyme preparations produced by the processes described above have been tested in regard to the proteolytic activity against hemoglobin at different pH values and different temperatures.

The activity was measured at pH 7, 7.5, 8, 9, 10, l 1, and 12 at 25 C, and expressed in percentage of maximum activity. The results are compiled in Table III below.

The activity was also measured at a constant pH value of 10.1 and varying temperatures, viz, 25, 37, 50, 60, 66, and 75 degrees of Celsius. The results of these measurements are compiled in Table IV below.

In general, the enzyme preparations produced according to the invention consist of a solid or liquid mixture of the proteolytic enzymes produced according to the invention and other components the amount and composition of which depend on the purpose and technical or scientific field within which the enzyme compositions are to be used. When the enzyme preparations or compositions according to the invention are in solid form they can consist of granules into which the enzymes are incorporated, for instance together with other enzymes or substances having other than enzymatic activity useful for the utility of the enzyme compositions. When the enzymes are not used in crystalline form, they may be accompanied by impurities of organic nature, such as proteins and carbohydrates from the culture medium. The enzyme powders may contain stabilizers. The enzyme compositions in liquid form can constitute solutions or suspensions which may contain stabilizers, if necessary.

Usually, the novel enzymes of the invention are used in small quantities. In view thereof the enzyme preparations or compositions for industrial use normally show an enzyme content not exceeding about 10 percent by weight. In some cases, however, the enzyme content may be considerably higher.

The novel enzymes according to the invention can for instance be used in washing compositions, dehairing compositions, in preparations for hydrolysis of proteins, in dish-washing compositions and as additives to septic tanks and installations for purifying sewage.

The enzyme powder produced from strain 8772 as described above has been used in washing experiments which were carried out while using EMPA test strips or swatches No. 1 16 soiled with blood, milk, and carbon black.

The detergent used had the following composition:

Dodecylbenzenesulphonate (50%) 20 g Nonylphenol, 9.5 E0 5 g Sodiumtripolyphosphate 30 g Sodiumcarbonate, anhydr. 15 g CMC (45%) 2 g Sodiumsulphate, anhydr. 28 g The washing conditions were as follows:

Hardness of water in German units 10 Fabric to water ratio 1:20 Time of experiment 30 minutes pH-value about 10 Detergent concentration 4.8 g per liter of washing solution Proteolytic activity 0.04 Anson unit measured at pH 7.5 per liter of washing solution Washing temperature 60 and 70 C The washing process carried out at 60 C was as follows:

By means of a pipette 20 ml of a solution prepared from the enzyme powder referred to above and showing a temperature of 20 C, a pH value of 11.2 and a proteolytic activity of 0.24 Anson unit per liter were added a 200 ml beaker. At zero time there were added ml detergent solution adjusted to to pH 1 1.3 and 68 C. The mixture was immediately transferred to a 200 ml long-necked Erlenmeyer flask placed on a shaking table and submerged in water kept at 60 C. Beforehand said flask contained 6 EMPA strips 1 l6 weighing in total 3.0 g. The shaking was continued during the washing period of 30 minutes. Then the washing solution was separated, and the pH value was measured after cooling. The test strips were rinsed and rubbed between the fingers under the hot water tap. All test strips were subjected to remission measurements in a Beckman spectrophotometer at 460 m;:..

In the experiments carried out at 70 C the detergent solution had a temperature of 80 C.

In addition to these experiments control experiments were carried out in which the enzyme solution was replaced by water.

Moreover, all the experiments were carried out while using an addition of 1 g of perborate (NaBO 4H 0 per liter of washing solution.

The results of these experiments are compiled in the following Table V TABLE V without perborate with perborate 60C C 60C Enzyme powder NClB 8772 54.7 49.2 41.5 I Control without j} enzyme 46.7 46,2 31.5

r: The remission values are average figures of measurements of each of the six test strips in each of the experiments.

What we claim is: r

l. A process of making proteolytic enzyme preparations;

UNITED STATES PATENT OFFICE} CERTIFICATE OF CORRECTION Patent No. 3 67H 6 13 Dated July 4 1972 lnven fl Knud Aunstrun et a1 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

'celumnz, line 3 change "79-78" to read "79-89-- Column 3, line 65, change Table III to read:

Table III pH-value 5O 75 85 100 NCIB 8772 33 v 5 1 67 71 85 100 um 70 80 100 NCTC M553 3M 52 6M 72 79 100 Signed and sealed this 1st day of May 1973.

(SILL XL) Attes't:

EDEJARD M. FLETCH R, JR. 7 ROBERT GOTTSCHALK A'ttesting Officer Commissioner of Patents QRM PC3-1050 (10-69) USCOMM'DC 6O376-P69 U.5. GOVERNMENT PRINTING OFFICE: 1969 07355-339 

2. The method according to claim 1 wherein the cultivation is carried out using a strain of bacterium of the genus Bacillus NCIB No.
 8772. 3. Proteolytic enzyme preparations produced by the method of claim
 1. 